【作者】 张成；

【导师】 黄科学；

【作者基本信息】 湖南师范大学 ， 微生物， 2010， 硕士

【摘要】 放线菌中的链霉菌和刺糖多孢菌是产生抗生素的重要来源。链霉菌Streptomyces lydicus产生的利迪链菌素(Streptolydigin)是一类重要的天然产物,为典型的Tetramic acid类抗生素,具有抗细菌活性、抗肿瘤活性、抗HIV蛋白酶活性。为了研究其生物合成途径中主要基因的功能,基于实验室已获得的基因簇,对聚酮合酶(PKS),两个细胞色素P450氧化酶(P450-1,P450-2),异柠檬酸脱氢酶(Idha),转氨酶(Adtf),2,3-己糖脱氢酶(Hdt)进行了基因中断,获得了同源单交换的中断突变株△PKS,△P450-1,△P450-2,△IDHA,△ADTF,△HDT。经过高效液相色谱(HPLC)和液相色谱和质谱耦连(LC-MS)检测证实中断突变株△PKS,△P450-1,△HDT不产生利迪链菌素或产生微量的利迪链菌素,说明这三个基因是利迪链菌素生物合成所必需的基因。然而中断突变株△P450-2,△IDHA,△ADTF仍然产生利迪链菌素,说明这些基因与利迪链菌素生物合成不相关或者菌体内存在其他基因,替补了这些基因的作用。这为理解利迪链菌素的生物合成提供了一定的基础,为提高利迪链菌素的产量和获得活性好的类似物提供了分子水平的依据。放线菌刺糖多孢菌,是一种商业杀虫剂Spinosad的产生菌,它能够产生在细胞内和细胞外具有脱氧核糖核苷酸酶活的酶,我们用PCR的方法从刺糖多孢菌中克隆了这些基因,并鉴定到两个脱氧核糖核苷酸酶基因(exoⅠ和exoⅡ),这两个酶在氨基酸序列上只有29%的同源性。本研究克隆了这两个基因,并在大肠杆菌中进行表达,纯化后对其进行了生化特征的研究。ExoⅠ在大肠杆菌中是以包涵体的形式表达的,通过复性后测定其活性。ExoⅡ通过低温诱导得到可溶性表达。这两个酶都显示了对单链DNA和双链DNA的酶切活性,但它们都需要二价阳离子(Mg2+,Ca2+)发挥活性,并且他们的活性都会受到螯合剂的抑制。这为研究脱氧核糖核苷酸内切酶在刺糖多孢菌发酵过程中所起的作用提供了体外的数据。更多还原

【Abstract】 Streptomyces and Saccharopolyspora, two actinomycetes, are important sources for producing antibiotics. Streptolydigin, an important natural secondary metabolite produced by Streptomyces lydicus, which belongs to classic tetramic acid, shows antimicrobic, antitumor and anti-HIV activity. In order to study the roles of genes in the streptolydigin biosynthesis pathway, based on the previous data in our lab, the polyketide synthase gene(PKS), two cytochrome P450 oxygenase gene (P450-1, P450-2), the isotratic dehydratase gene (Idha), the like amino transferase gene(Adtf), the 2,3-glucose dehydratase gene(Hdt) were inactivated by disrupting, and the disrupted mutants△PKS,△P450-1,△P450-2,△IDHA,△ADTF,△HDT were obtained by single crossover. No streptolydigin or little production was detected in the mutant strains (△PKS,△P450-1, AHDT) cultured in the fermentation broth, the results confirmed that the PKS, the cytochrome P450 oxygenase-1, Hdt were essential for streptolydigin biosynthesis. While streptolydigin production was still detected in the mutant strains△P450-2, AIDHA, AADTF, indicates that these genes may be not involved in the biosynthesis pathway of streptolydigin or some alternative genes exist in the stain. The research provides basis for understanding the biosynthesis pathway of streptolydigin, improving the yield of streptolydigin, and obtaining derives which show better activity. Saccharopolyspora spinosa, a commercial insecticide spinosad producer, produces intra-and extracellular enzymes with deoxyribonuclease activities. PCR were used to clone these deoxyribonuclelases and two deoxyribonuclease genes(exo I and exo II) were identified from Saccharopolyspora spinosa. Exo I and Exo II show only 29% amino acids sequence identities. These two genes were amplified and expressed in Escherichia coli, purified to homogeneity and characterized. The Exo I was expressed and refolded from inclusion bodies. The Exo II was expressed in soluble fraction when induced at low temperature. Both enzymes showed activities either on ssDNA or dsDNA. They required a divalent cation (Mg2+, Ca2+) and its activity was inhibited by chelating agents. The results provide datas in vitro for studying the roles of the two deoxyribonucleases in the process of fermentation of Saccharopolyspora spinosa.更多还原